Note: Descriptions are shown in the official language in which they were submitted.
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PLANT ION CHANNELS AND METHODS
REFERENCES TO RELATED APPLICATIONS
This application claims the benefit of U.S. Provisional Application No.
60/306,819, filed July 20, 2001, which is hereby incorporated by reference
herein
in its entirety.
BACKGROUND OF THE INVENTION
The amino acid'y aminobutyric acid (GAGA) is the major neurotransmitter
in the mammalian central nervous system. Such neurotransmitters generally
function in regulating the conductance of ions across neuronal membranes,
typically in regulating influx of ions into a cell. For example, GABA is
considered
an inhibitory neurotransmitter that acts to inhibit synaptic transmission in
both
vertebrate and invertebrate nervous systems. As another example, glutamate is
an
excitatory neurotransmitter that depolarizes the postsynaptic membrane and
acts to
promote synaptic transmission. Both GABA and glutamate affect synaptic
transmission by binding to their respective receptors, also known as ligand-
gated
ion channels.
These ligand-gated ion channels are present in neurons of insects and
animals. Three general classes of GABA receptors, denoted GABAA, GABAB and
GABA~, are present in animal neurons. GABA receptors have been implicated in
mediating anxiety, seizures, cognitive function, addictive disorders, sleep
disorders
and other disorders of the central nervous system. GABA receptors are the
target
of many pharmaceutical preparations that act on the central nervous system,
including barbiturates and benzodiazepines, and thus have therapeutic value.
Furthermore, compounds that affect the function of insect GABA receptors are
commercially useful as insecticides.
3o GABA receptors have been found in insects and in the animal kingdom.
Recently, proteins, or other molecules through which GABA may act, that are
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2
expected to function as GABA receptors have been discovered in the plant
kingdom (U.S. Patent Application Serial No. 09/517,438, filed March 2, 2000).
GABA has been shown to exert certain beneficial effects on plants. For
example, GABA has been shown to increase plant growth and productivity as
shown in U.S. Patent No. 5,439,873 to Kinnersley. Moreover, such beneficial
effects have been increased when GABA is applied to plants along with a
readily
metabolized source of carbon, such as succinic acid (U.S. Patent No.
5,604,177).
Moreover, GABA has been found to increase fertilizer efficiency when
administered with glutamic acid as described in U.S. Patent No. 5,840,656 to
Kinnersley et al.
The mechanism of the above-described beneficial results of GABA in
plants has not yet been confirmed. A better understanding of the mechanism of
GABA-mediated plant growth and productivity and other mechanisms in which
GABA is involved is expected to lead to further methods for improving plant
growth, productivity, and other beneficial effects.
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SUMMARY OF THE INVENTION
The present invention relates to the new discovery that plants respond to
compounds known to act on animal mitochondria) GABA receptor proteins, and
the related discovery that plants express receptor proteins that respond to
these
compounds. In this regard, the invention provides nucleotide sequences that
have
been discovered in plants that are expected to encode benzodiazepine or
benzodiazepine-like receptor proteins having significant sensitivity to
benzodiazepines. Based upon the data presented herein, such proteins are
expected
to function as modulators of GABA action and, in particular, as ion channels,
such
as ligand-gated ion channels. Furthermore, the proteins are expected to
participate
in stress-related physiological response of plants, and incorporation of
nucleic acid
molecules encoding the proteins into a plant is expected to enhance the
plant's
ability to withstand stresses. Accordingly, the present invention provides
purified
plant proteins, including recombinant proteins, nucleotide sequences encoding
the
proteins and methods of using the nucleotide sequences and proteins.
In one aspect of the invention, methods of transforming a plant are
provided. In one form of the invention, a method includes introducing into a
plant
cell a nucleic acid molecule encoding a plant protein described herein.
In a second aspect of the invention, methods of treating a plant are provided
that include providing a plant having an introduced nucleotide sequence
encoding a
plant protein described herein and treating the plant with an effective amount
of
GABA. In alternative embodiments, the plant is treated with a composition
including GABA and a GABA agonist or is treated only with a GABA antagonist
or GABA agonist. In a further embodiment, a plant is treated with agonists or
antagonists of animal benzodiazepine receptors and including agonists or
antagonists of peripheral benzodiazepine receptors in animals.
In a third aspect of the invention, methods of regulating plant metabolism
are provided that include utilizing antisense DNA or RNA to reduce formation
of a
plant protein or RNA transcript, such as an mRNA transcript. In one
embodiment,
3o the method includes introducing into a plant cell an antisense nucleic acid
molecule having a nucleotide sequence that is complementary to a coding
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4
nucleotide sequence described herein, or a portion thereof. Alternatively, the
antisense nucleic acid molecule includes a nucleotide sequence complementary
to
an RNA sequence, preferably a mRNA sequence, transcribed from a sequence
described herein. The antisense nucleotide sequence hybridizes to nucleic
acid,
including either the template strand or the RNA transcript, of the plant to
reduce
formation of a plant protein described herein.
In a fourth aspect of the invention, methods of identifying potential plant
receptors are provided that include hybridizing to plant nucleic acid a probe
having
a nucleotide sequence encoding the proteins described herein or a portion
thereof.
In a fifth aspect of the invention, methods of expressing plant proteins
described herein are provided. In one embodiment, a method includes
introducing
into a host cell a nucleotide sequence encoding a plant receptor protein as
described herein and culturing under conditions to achieve expression of the
receptor protein.
In further embodiments, isolated nucleic acid molecules, including
recombinant nucleic acid molecules, are provided that include nucleotide
sequences encoding plant proteins as described herein. Plant host cells and
transgenic plants are also provided that include nucleotide sequences encoding
a
plant protein described herein. The molecules, plant cells and transgenic
plants
further may include a foreign promoter sequence operably linked to a terminal
5'
end of the plant nucleotide sequences described herein.
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BRIEF DESCRIPTION OF THE FIGURES
Although the characteristic features of this invention will be particularly
pointed out in the claims, the invention itself, and the manner in which it
may be
made and used, may be better understood by referring to the following
description
taken in connection with the accompanying figures forming a part hereof.
FIG. 1 depicts a schematic showing the proposed roles of GABA in plant
stress responses (hypothetical pathways by which GABA may function as a
cellular barometer and transducer of environmental stress signals).
to FIG. 2 depicts a graph showing the effect of cyclosporin A on GABA-
mediated growth promotion in duckweed as more fully described in Example 1.
FIG. 3 depicts a graph showing the effect of spermine on GABA-mediated
growth promotion in duckweed as more fully described in Example 1.
FIG. 4 depicts a graph showing the effect of quinine on GABA-mediated
growth promotion in duckweed as more fully described in Example 1.
FIG. 5 depicts a graph showing the effect of diazepam and PI~11195
(isoquinoline carboxamide) on GABA-mediated growth promotion in duckweed as
more fully described in Example 1.
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6
DESCRIPTION OF THE PREFERRED EMBODIMENTS
For the purposes of promoting an understanding of the principles of the
invention, reference will now be made to preferred embodiments and specific
language will be used to describe the same. It will nevertheless be understood
that
no limitation of the scope of the invention is thereby intended, such
alterations and
further modifications of the invention, and such further applications of the
principles of the invention as described herein, being contemplated as would
normally occur to one skilled in the art to which the invention relates.
to The present invention relates to the discovery that plants respond to
compounds that are known to act on animal mitochondrial GABA receptor
proteins, and the related discovery that plants express receptor proteins that
respond to these compounds. The invention further relates to the discovery of
a
nucleotide sequence in Arabidopsis thalia~ca that is expected to encode a
plant
15 benzodiazepine, and/or benzodiazepine-like, receptor protein (hereinafter
referred
to collectively as "receptor protein"). The invention also relates to
nucleotide
sequences that encode analogous receptor proteins in other species and that
exhibit
similar functionality and have sequence identity to the exemplary Arabidopsis
thaliana sequences set forth herein. Accordingly, the present invention
provides
2o purified receptor proteins and isolated nucleic acid molecules comprising
nucleotide sequences encoding plant receptor proteins. Recombinant nucleic
acid
molecules, plant host cells and transgenic plants are also provided that
include the
nucleotide sequences encoding the plant receptor proteins. In other aspects of
the
invention, methods of expressing a receptor protein, and methods of using the
25 nucleotide and amino acid sequences described herein are also provided.
In one aspect of the invention, purified plant benzodiazepine or
benzodiazepine-like receptor proteins are provided. While it is not intended
that
the invention be limited by any theory whereby it achieves its advantageous
result,
it is believed that plant receptor proteins described herein function as ion
channel
3o proteins, such as ligand-gated ion-channel proteins in plants, and
therefore have the
ability to regulate cellular ion influx and/or transport ions within a cell.
Candidate
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ions whose entry may be regulated include anions, such as chloride and canons,
such as calcium, sodium, and potassium. The receptors may, for example,
release
calcium ions from intracellular stores into the cytosol. In accordance with
this
aspect of the invention, receptor proteins are provided that are substantially
pure.
As used herein, "substantially pure" is intended to mean that the receptor
proteins
are at least about 95% free from other proteins with which they naturally
occur.
In one embodiment, an Arabidopsis thaliana receptor protein in accordance
with the invention has the amino acid sequence as set forth in SEQ ID N0:2.
Although the invention is described with reference to an Arabidopsis thaliana
to amino acid sequence, it is understood that the invention is not limited to
the
specific amino acid sequence set forth in SEQ ID N0:2. Skilled artisans will
recognize that, through the process of mutation and/or evolution, polypeptides
of
different lengths and having differing constituents, e.g., with amino acid
insertions,
substitutions, deletions, and the like, may arise that are related to, or
sufficiently
15 similar to, a sequence set forth herein by virtue of amino acid sequence
homology
and advantageous functionality as described herein. The terms "benzodiazepine
receptor protein" and "benzodiazepine-like receptor protein" are used herein
to
refer generally to a protein having the features described herein, one example
of
which is a polypeptide having the amino acid sequence set forth in SEQ ID NO:
2.
20 Further included within this definition, and in the scope of the invention,
are
variants of the polypeptide which have the structural features and exhibit the
functionality described herein.
It is well known that plants of a wide variety of species commonly express
and utilize homologous proteins, which include the insertions, substitutions
and/or
25 deletions discussed above, and yet which effectively provide similar
function. For
example, an amino acid sequence isolated from another species may differ to a
certain degree from the sequence set forth in SEQ >D NO: 2, and yet be readily
recognizable by a person of ordinary skill in the art as an analogous protein
expected to have similar functionality. Amino acid sequences comprising such
30 variations that have similar functionality and that have a stated degree of
identity
are included within the scope of the present invention. Although it is not
intended
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that the present invention be limited by any theory by which it achieves its
advantageous result, it is believed that the identity between amino acid
sequences
that is necessary to maintain proper functionality is related to maintenance
of the
tertiary structure of the polypeptide such that specific interactive sequences
will be
properly located and will have the desired activity. It is contemplated that a
polypeptide including these interactive sequences in proper spatial context
will
have good activity, even where alterations exist in other portions thereof. In
this
regard, a variant of the protein described herein is expected to be
functionally
similar to that set forth in SEQ m NO: 2, for example, if it includes amino
acids
which are conserved among a variety of plant species or if it includes non-
conserved amino acids which exist at a given location in another plant species
that
expresses a protein as described herein.
Another manner in which similarity rnay exist between two amino acid
sequences is where a given amino acid of one group (such as a non-polar amino
acid, an uncharged polar amino acid, a charged polar acidic amino acid or a
charged polar basic amino acid) is substituted with another amino acid from
the
same amino acid group. For example, it is known that the uncharged polar amino
acid serine may commonly be substituted with the uncharged polar amino acid
threonine in a polypeptide without substantially altering the functionality of
the
polypeptide. Whether a given substitution will affect the functionality of the
enzyme may be determined without undue experimentation using synthetic
techniques and screening assays known in the art, including screens employing
methods set forth in the Examples below.
In one embodiment, the invention provides amino acid sequences that have
at least about 60% identity to the amino acid sequence set forth in SEQ >D NO:
2
and that exhibit similar functionality as the amino acid sequence set forth in
SEQ
ll~ NO: 2. In another embodiment, the invention provides a receptor protein
having an amino acid sequence that has at least about 70% identity to the
amino
acid sequence set forth in SEQ m NO: 2 and that exhibits similar functionality
as
the amino acid sequence set forth in SEQ ID NO: 2. In another embodiment, the
invention provides a receptor protein having an amino acid sequence that has
at
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least about 80% identity to the amino acid sequence set forth in SEQ ID NO: 2
and
that exhibits similar functionality as the amino acid sequence set forth in
SEQ ID
NO: 2. In another embodiment, the invention provides a receptor protein having
an amino acid sequence that has at least about 90% identity to the amino acid
sequence set forth in SEQ ID NO: 2 and that exhibits similar functionality as
the
amino acid sequence set forth in SEQ ID NO: 2.
Percent identity may be determined, for example, by comparing sequence
information using the MacVector computer program, version 6Ø1, available
from
Oxford Molecular Group, Inc. (Beaverton, OR). Briefly, the MacVector program
defines identity as the number of identical aligned symbols (i.e., nucleotides
or
amino acids), divided by the total number of symbols in the shorter of the two
sequences. The program may be used to determine percent identity over the
entire
length of the proteins being compaxed. Preferred default parameters for the
MacVector program include: for pairwise alignment: (1) matrix = BLOSUM30;
(2) Alignment speed - fast; (3) Ktuple = 1; (4) Gap penalty = l; Top diagonals
= 5;
Window size = 5; for multiple alignment: matrix = BLOSUM series, open gap
penalty = 10; extended gap penalty = 0.1, delay divergent = 40%; protein gap
parameters: Gap separation distance = 8; residue-specific penalties = yes or
on;
hydrophilic residues = GPSNDQEKR.
2o In another aspect of the invention, isolated nucleic acid molecules are
provided that encode a protein as described herein. In one embodiment, the
invention provides a nucleotide sequence, originally isolated from Arabidopsis
thaliana, as set forth in SEQ ID NO: 1. It is to be understood that sequences
complementary to the specific sequence shown therein are also encompassed in
the
invention. In one form of the invention, an isolated nucleic acid molecule is
provided that has a nucleotide sequence encoding a protein having an amino
acid
sequence having at least about 60% identity to the amino acid sequence set
forth in
SEQ ID NO: 2 and that exhibits similar functionality as the amino acid
sequence
set forth in SEQ ID NO: 2. In another embodiment, the invention provides an
3o isolated nucleic acid molecule that has a nucleotide sequence encoding a
protein
having an amino acid sequence having at least about 70% identity to the amino
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acid sequence set forth in SEQ ID NO: 2 and that exhibits similar
functionality as
the amino acid sequence set forth in SEQ ID NO: 2. In another embodiment, the
invention provides an isolated nucleic acid molecule that has a nucleotide
sequence
encoding a protein having an amino acid sequence having at least about 80%
5 identity to the amino acid sequence set forth in SEQ ID NO: 2 and that
exhibits
similar functionality as the amino acid sequence set forth in SEQ ID NO: 2. In
another embodiment, the invention provides an isolated nucleic acid molecule
that
has a nucleotide sequence encoding a protein having an amino acid sequence
having at least about 90% identity to the amino acid sequence set forth in SEQ
D7
to NO: 2 and that exhibits similar functionality as the amino acid sequence
set forth
in SEQ ID NO: 2.
It is not intended that the present invention be limited to these exemplary
nucleotide sequences, but include sequences having substantial similarity
thereto
and sequences which encode variant forms of the plant receptor proteins
described
herein as discussed above and as further discussed below.
The term "isolated nucleic acid," as used herein, is intended to refer to
nucleic acid that is not in its native environment. For example, this term
refers to
nucleic acid that is separated from other contaminants that naturally
accompany it,
such as proteins, lipids and other nucleic acid sequences. The term includes
nucleic
2o acid that has been removed or purified from its naturally occurring
environment or
clone library, and further includes recombinant or cloned nucleic acid
isolates and
chemically synthesized nucleic acid.
The term "nucleotide sequence," as used herein, is intended to refer to a
natural or synthetic linear and sequential array of nucleotides and/or
nucleosides,
including deoxyribonucleic acid, ribonucleic acid, and derivatives thereof.
The
terms "encoding" and "coding" refer to the process by which a nucleotide
sequence, through the mechanisms of transcription and translation, provides
the
information to a cell from which a series of amino acids can be assembled into
a
specific amino acid sequence to produce a functional polypeptide, such as, for
3o example, an active enzyme or other protein that has a specific function.
The
process of encoding a specific amino acid sequence may involve DNA sequences
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having one or more base changes (i.e., insertions, deletions, substitutions)
that do
not cause a change in the encoded amino acid, or which involve base changes
which may alter one or more amino acids, but do not eliminate the functional
properties of the polypeptide encoded by the DNA sequence.
It is therefore understood that the invention encompasses more than the
specific exemplary nucleotide sequence set forth in SEQ ID NO: 1. For example,
nucleic acid sequences encoding variant amino acid sequences, as discussed
above,
are within the scope of the invention. Modifications to a sequence, such as
deletions, insertions, or substitutions in the sequence, which produce
"silent"
changes that do not substantially affect the functional properties of the
resulting
polypeptide molecule are expressly contemplated by the present invention. Fox
example, it is understood that alterations in a nucleotide sequence which
reflect the
degeneracy of the genetic code, or which result in the production of a
chemically
equivalent amino acid at a given site, are contemplated. Thus, a codon for the
amino acid alanine, a hydrophobic amino acid, may be substituted by a codon
encoding another less hydrophobic residue, such as glycine, or a more
hydrophobic
residue, such as valine, leucine, or isoleucine. Similarly, changes which
result in
substitution of one negatively charged residue for another, such as aspartic
acid for
glutamic acid, or one positively charged residue for another, such as lysine
for
arginine, are also contemplated by the present invention when the nucleotide
sequence having such changes is expected to produce a biologically equivalent
product.
Nucleotide changes which result in alteration of the N-terminal and C-
terminal portions of the encoded polypeptide molecule would also not generally
be
expected to alter the activity of the polypeptide. In some cases, it may in
fact be
desirable to make mutations in the sequence in order to study the effect of
alteration on the biological activity of the polypeptide. Each of the proposed
modifications is well within the routine skill in the art.
In one preferred embodiment, the present invention provides a nucleotide
sequence that has substantial similarity to the entire sequence set forth in
SEQ ID
NO: 1, and variants described herein. The term "substantial similarity" is
used
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herein with respect to a nucleotide sequence to designate that the nucleotide
sequence has a sequence sufficiently similar to a reference nucleotide
sequence
that it will hybridize therewith under moderately stringent conditions. This
method
of determining similarity is well known in the art to which the invention
pertains.
Briefly, moderately stringent conditions are defined in Sambrook et al.,
Molecular
Cloning: A Laboratory Manual, 2nd ed. Vol. 1, pp. 101-104, Cold Spring Harbor
Laboratory Press (1989) as including the use of a prewashing solution of 5X
SSC
(a sodium chloridelsodium citrate solution), 0.5% sodium dodecyl sulfate
(SDS),
1.0 mM ethylene diaminetetraacetic acid (EDTA) (pH 8.0) and hybridization and
to washing conditions of 55°C, 5x SSC. A further requirement of the
inventive
polynucleotide is that it must encode a polypeptide having similar
functionality to
the plant proteins described herein.
In yet another embodiment, nucleotide sequences having selected percent
identities to specified regions of the nucleotide sequence set forth in SEQ ID
NO: 1
are provided. In one form of the invention, nucleotide sequences are provided
that
have at least about 50% identity to a nucleotide sequence of substantial
length
within the nucleotide set forth in SEQ ID NO: 1. In another embodiment, the
invention provides a nucleotide sequence that has at least about 60% identity
to a
nucleotide sequence of substantial length within the nucleotide set forth in
SEQ ID
2o NO: 1. In another embodiment, the invention provides a nucleotide sequence
that
has at least about 70% identity to a nucleotide sequence of substantial length
within the nucleotide set forth in SEQ m NO: 1. In another embodiment, the
invention provides a nucleotide sequence that has at least about 80% identity
to a
nucleotide sequence of substantial length within the nucleotide set forth in
SEQ ID
NO: 1. In another embodiment, the invention provides a nucleotide sequence
that
has at least about 90% identity to a nucleotide sequence of substantial length
within the nucleotide set forth in SEQ ID NO: 1.
In one embodiment, "substantial length" refers to a length of at least about
50
nucleotides. In another embodiment, the substantial length is a length of at
least
3o about 100 nucleotides. In another embodiment, the substantial length is a
length of at
least about 200 nucleotides. In another embodiment, the substantial length is
a length
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of at least about 300 nucleotides. In another embodiment, the substantial
length is a
length of at least about 400 nucleotides. In another embodiment, the
substantial
length is a length of at least about 500 nucleotides. In another embodiment,
the
substantial length is the entire sequence set forth in SEQ >D NO: 1.
The percent identity may be determined, for example, by comparing
sequence information using the MacVector program, as described above with
reference to amino acid identity. Preferred default parameters include: (1)
for
pairwise alignment parameters: (a) I~tuple = 1; (b) Gap penalty = 1; (c)
Window
size = 4; and (2) for multiple alignment parameters: (a) Open gap penalty =
10; (b)
Extended gap penalty = 5; (c) Delay divergent = 40%o; and (d) transitions =
weighted. A further requirement of a nucleotide sequence in accordance with
the
invention is that it encodes a protein that functions as described herein.
A suitable DNA sequence in accordance with the invention may be
obtained by cloning techniques using cDNA or genomic libraries of Arabidopsis
thaliaraa or other species, which are available commercially or which may be
constructed using standard methods known in the art. Suitable nucleotide
sequences may be isolated from DNA libraries obtained from a wide variety of
species by means of nucleic acid hybridization or polymerase chain reaction
(PCR)
procedures, using as probes or primers nucleotide sequences selected in
accordance
with the invention, such as that set forth in SEQ ID NO:1, nucleotide
sequences
having substantial similarity thereto, or portions thereof. In preferred forms
of the
invention, the nucleotide sequences provided herein are cDNA sequences.
Alternately, a suitable sequence may be made by techniques that are well
known in the art. For example, nucleic acid sequences encoding a plant protein
described herein may be constructed by recombinant DNA technology, for
example, by cutting or splicing nucleic acids using restriction enzymes and
DNA
ligase. Furthermore, nucleic acid sequences may be constructed using chemical
synthesis, such as solid-phase phosphoramidate technology, or PCR. PCR may
also be used to increase the quantity of nucleic acid produced. Moreover, if
the
3o particular nucleic acid sequence is of a length which makes chemical
synthesis of
the entire length impractical, the sequence may be broken up into smaller
segments
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which may be synthesized and ligated together to form the entire desired
sequence
by methods known in the art.
In a further aspect of the invention, recombinant nucleic acid molecules, or
recombinant vectors, are provided. In one embodiment, a nucleic acid molecule
is
provided that includes a nucleotide sequence as described herein. The protein
encoded by the nucleotide sequence has the amino acid sequence set forth in
SEQ
m N0:2, or variants thereof as described above.
A wide variety of vectors are known that have use in the invention. For
example, various plasmid and phage vectors are known that are ideally suited
for
to use in the invention, including ,Zap and pBluescript. In preferred
embodiments,
the vector may be a T-DNA vector. Representative T-DNA vector systems are
discussed in the following publications: An et al., (1986) EMBO J. 4:277;
Herrera-
Estrella et al., (1983) EMBO J. 2:987; Herrera-Estrella et al., (1985) in
Plant
Genetic Engineering, New York: Cambridge University Press, p. 63.
In one embodiment, the desired recombinant vector may be constructed by
ligating DNA linker sequences to the 5' and 3' ends of the desired nucleotide
insert, cleaving the insert with a restriction enzyme that specifically
recognizes
sequences present in the linker sequences and the desired vector, cleaving the
vector with the same restriction enzyme, mixing the cleaved vector with the
2o cleaved insert and using DNA ligase to incorporate the insert into the
vector as
known in the art.
The vectors may include other nucleotide sequences, such as those
encoding selectable markers, including those for antibiotic resistance or
color
selection. The vectors also preferably include a promoter nucleotide sequence.
The desired nucleic acid insert is preferably operably linked to the promoter.
A
nucleic acid is "operably linked" to another nucleic acid sequence, such as a
promoter sequence, when it is placed in a specific functional relationship
with the
other nucleic acid sequence. The functional relationship between a promoter
and a
desired nucleic acid insert typically involves the nucleic acid and the
promoter
sequences being contiguous such that transcription of the nucleic acid
sequence
will be facilitated. Two nucleic acid sequences are further said to be
operably
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linked if the nature of the linkage between the two sequences does not (1)
result in
the introduction of a frame-shift-mutation; (2) interfere with the ability of
the
promoter region sequence to direct the transcription of the desired nucleotide
sequence, or (3) interfere with the ability of the desired nucleotide sequence
to be
5 transcribed by the promoter sequence region. Typically, the promoter element
is
generally upstream (i.e., at the 5' end) of the nucleic acid insert coding
sequence.
A wide variety of promoters are known in the art, including cell-specific
promoters, inducible promoters, and constitutive promoters. Such promoters
that
direct transcription in plants cells may be used. The promoters may be of
viral,
to bacterial or eukaryotic origin, including those from plants and plant
viruses. For
example, in certain preferred embodiments, the promoter may be of viral
origin,
including a cauliflower mosaic virus promoter (CaMV), such as CaMV 35S or
195, a figwort mosaic virus promoter (FMV 35S), or the coat protein promoter
of
tobacco mosaic virus (TMV). The promoter may further be, for example, a
15 promoter for the small subunit of ribulose-1,3-diphosphate caxboxylase.
Promoters
of bacterial origin include the octopine synthase promoter, the nopaline
synthase
promoter and other promoters derived from native Ti plasmids as discussed in
Herrera-Estrella et al., Nature, 303:209-213 (1983).
The promoter may further be one that responds to various forms of
2o environmental stresses, or other stimuli. For example, the promoter may be
one
induced by abiotic stresses such as wounding, cold, dessication, ultraviolet-B
[van
Der Krol et al. (1999) Plant Physiol. 121:1153-1162], heat shock [Shinmyo et
al.,
(1998) Biotechnol. Bioeng. 58:329-332] or other heat stress, drought stress or
water stress. The promoter may further be one induced by biotic stresses
including
pathogen stress, such as stress induced by a virus [Sohal et al. (1999) Plant
Mol.
Biol. 41:75-87] or fungi [Eulgem (1999) EMBO. J. 18:4689-4699], stresses
induced as part of the plant defense pathway [Lebel (1998) Plant J. 16:223-
233] or
by other environmental signals, such as light [Ngai et al. (1997) Plant J.
12:1021-
1034; Sohal et al. (1999) Plant Mol. Biol. 41:75-87], carbon dioxide [I~ucho
et al.
( 1999) Plant Physiol 121:1329-1338], hormones or other signaling molecules
such
as auxin, hydrogen peroxide and salicylic acid [Chen and Singh (1999) Plant J.
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16
19:667-677], sugars and gibberellin [Lu et al. ( 1998) J. Biol. Chem.
273:10120-
10131] or abscissic acid and ethylene [Leubner-Metzger et al. (1998)
PlaratMol.
Biol. 38:785-795].
The promoters may further be selected such that they require activation by
other elements known in the art, so that production of the protein encoded by
the
nucleic acid sequence insert may be regulated as desired. In one embodiment,
the
promoter is a foreign promoter. A "foreign promoter" is defined herein to mean
a
promoter other than the native, or natural, promoter that promotes
transcription of
a length of DNA.
The vectors may further include other regulatory elements, such as
enhancer sequences, which cooperate with the promoter to achieve transcription
of
the nucleic acid insert coding sequence. By "enhancer" is meant nucleotide
sequence elements that can stimulate promoter activity in a cell, such as a
plant
host cell. The vectors may further include 3' regulatory sequence elements
known
in the art, such as those, for example, that increase the stability of the RNA
transcribed.
Moreover, the vectors may include another nucleotide sequence insert that
encodes a peptide or polypeptide used as a tag to aid in purification of the
desired
protein encoded by the desired nucleotide sequence or that encodes another
functional protein. With respect to inclusion of a tag, the additional
nucleotide
sequence can be positioned in the vector such that a fusion, or chimeric,
protein is
obtained. For example, a protein described herein may be produced having at
its
C-terminal end linker amino acids, as known in the art, joined to the other
protein
that acts as a tag. After purification procedures known to the skilled
artisan, the
additional amino acid sequence is cleaved with an appropriate enzyme. The
protein
may then be isolated from the other proteins, or fragments thereof, by methods
known in the art.
In another embodiment, a vector includes a second nucleotide sequence that
encodes another functional protein, such as, for example, a plant GAD enzyme,
as
3o described in the inventors' copending U.S. patent application, Serial No.
10/006,852, which is hereby incorporated herein by reference. Alternatively,
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plants can be transformed in accordance with the invention with two different
vectors, one including a DNA construct for expression of a GAD enzyme, by way
of example, and the other for expression of a plant receptor protein as
described
herein. It is expected that overexpression of a GAD enzyme and a receptor
protein
in a plant will result in a plant with excellent features, such as, for
example,
enhanced stress resistance.
The inventive recombinant vectors may be used to transform a host cell.
Accordingly, methods of transforming a cell or a plant are provided that
include
introducing into a plant cell a nucleic acid molecule having an inventive
nucleotide
to sequence. A wide variety of methods of transforming a cell or a plant are
well
known in the art, and may be found in references including, for example,
Maniatis
et al., Molecular Cloning: A Laboratory Manual, Cold Springs Laboratory, Cold
Springs Harbor, New York ( 1982) and Current Protocols in Molecular Biology,
John Wiley and Sons, edited by Ausubel et al. (1988). Plant gene transfer
15 techniques may also be found in references including Fromm et al., (1985)
Proc.
Natl. Acad. Sci. USA , 82:5824-5828 (lipofection); Crossway et al., (1986)
Mol.
Gen. Genet. 202:179 (microinjection); Hooykaas-Van Slogtern et al., (1984)
Nature 311:763-764)(T-DNA mediated transformation of monocots); Rogers et al.,
( 1986) Methods Enzymol. 118:627-641 (T-DNA mediated transformation of
20 dicots); Bevan et al., (1982) Ann. Rev. Genet. 16:357-384) (T-DNA mediated
transformation of dicots); Klein et al., (1988) Proc. Natl. Acad. Sci USA
85:4305-
4309 (microprojectile bombardment); and Fromm et al., Nature (1986) 319:791-
793 (electroporation). The introduced polynucleotide, in an appropriate
vector, is
advantageously integrated into the plant genome, but may remain episomal in
other
25 forms of the invention. Once the desired nucleic acid has been introduced
into a
host cell or a host plant, the host cell expresses the protein. Accordingly,
in yet
another aspect of the invention, a host cell is provided that includes the
inventive
recombinant vectors described above.
A wide variety of host cells may be used in the invention, including
3o prokaryotic and eukaryotic host cells. Preferred host cells are eukaryotic
and are
further preferably plant cells, such as, for example, those derived from
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monocotyledons, such as duckweed, corn, turf (including rye grass, Bermuda
grass, Blue grass, Fescue), dicotyledons, including lettuce, cereals such as
wheat,
crucifers (such as rapeseed, radishes and cabbage), solanaceae (including
green
peppers, potatoes and tomatoes), and legumes such as soybeans and bush beans.
In
a further aspect of the invention, the host cells may be cultured as known in
the art
to produce a transgenic plant. A transformed plant can be made, for example,
by
transforming a cell, tissue or organ from a host plant with an inventive
nucleic acid
molecule; selecting a transformed cell, cell callus, somatic embryo, or seed
which
contains the nucleic acid molecule; regenerating a whole plant from the
selected
transformed cell, cell callus, somatic embryo, or seed; and selecting a
regenerated
whole plant that expresses the nucleotide sequence.
In another aspect of the invention, methods of identifying plant proteins,
such as those expected to be benzodiazepine or benzodiazepine-like receptors,
are
provided. In these methods, nucleotide sequences described above, or portions
thereof, are used as probes to locate other, similar nucleotide sequences that
may
encode other benzodiazepine or benzodiazepine-like receptors. General methods
for screening for selected nucleotide sequences in a DNA or RNA sample are
known to the art. For example, DNA may be isolated from selected plants,
treated
with various restrictions enzymes and analyzed by Southern blotting techniques
utilizing a radioactively or fluorescently-labeled probe of interest. RNA
fragments
may be similarly analyzed by Northern blotting techniques. Alternatively,
commercially available cDNA or genomic libraries may be screened.
In one embodiment, a nucleic acid molecule used as a probe has a
nucleotide sequence having at least about 60% identity to a nucleotide
sequence
having a length of about 25 to about 100 nucleotides within the nucleotide
sequence set forth in SEQ ID NO:1. In another embodiment, a nucleic acid
molecule used as a probe has a nucleotide sequence having at least about 60%
identity to a nucleotide sequence having a length of about 25 to about 400
nucleotides within the nucleotide sequence set forth in SEQ ID NO:1. In
another
3o embodiment, a nucleic acid molecule used as a probe has a nucleotide
sequence
having at least about 60% identity to a nucleotide sequence having a length of
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about 25 to about 500 nucleotides within the nucleotide sequence set forth in
SEQ
ID NO:1. In another embodiment, the probe has a nucleotide sequence having at
least about 60% identity to the entire length of nucleotides set forth in SEQ
>D
NO:1. In another embodiment, the probe has a nucleotide sequence having at
least
about 70% identity to the length of nucleotides indicated directly above. In
another embodiment, the probe has a nucleotide sequence having at least about
SO% identity to the length of nucleotides indicated directly above. In another
embodiment, the probe has a nucleotide sequence having at least about 90%
identity to the length of nucleotides indicated directly above. The probe may
be
radioactively labeled at its 5'end, for example, with polynucleotide kinase
and 32P
and hybridized to the isolated nucleic acid fragments.
In another aspect of the invention, methods of treating a plant are provided.
In one embodiment, a method includes providing a plant having an introduced
nucleic acid molecule described herein, wherein the plant expresses the
encoded
receptor protein, and treating the plant with an effective amount of GABA.
Such
treating of the plant is expected to advantageously stimulate growth of the
plant, as
well as provide other beneficial results, including reducing the effects of
plant stress.
In one embodiment, transgenic plants are prepared as described above and
treated with an effective amount of GABA. As used herein, "effective amount"
refers to an amount of GABA that will provide one or more advantages to the
plant, such as, for example, stimulation of plant growth and/or reduction of
plant
stress. The amount may vary depending upon a wide vaxiety of factors,
including,
for example, the particular advantage provided to the plant, the number of
introduced nucleotide sequences expressed, the type of plant, the number of
plants
treated and the environmental conditions. In one embodiment, plants are
treated
with about 1 ppm to about 24,000 ppm GABA [about 0.013 ozlacre (oz/A) to
about 20 lbs/A] [about 0.93 g/hectare (g/ha) to about 22 kg/ha]. In another
embodiment, plants are treated with about 1 ppm to about 12,000 ppm GABA
[about 0.013 oz/A to about 10 lbs/A] [about 0.93 g/ha to about 11 kg/ha]. In
3o another embodiment, plants are treated with about 1 ppm to about 7,500 ppm
GABA [about 0.013 ozlA to about 6.3 lbs/A] [about 0.93 g/ha to about 7.1
kg/ha].
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In another embodiment, plants are treated with about 1 ppm to about 5,000 ppm
GABA [about 0.013 oz/A to about 4.2 lbs/A] [about 0.93 g/ha to about 4.8
kg/ha].
With respect to plant growth stimulation, concentrations of about 1 ppm to
about
5,000 ppm, as described in U.S. Patent No. 5,439,873 to Kinnersley, may be
advantageously employed. When reduction of plant stress is desired,
concentrations of GABA of from about 1 ppm to about 2,500 ppm [about 0.013
oz/A to about 2.1 lbs/A] [about 0.93 g/ha to about 2.4 kg/ha] may be
advantageously employed. About 150-600 ppm [about 1/8 lb/A to about 1/2 lb/A]
[about 0.14 kg/ha to about 0.56 kg/ha] employed in one embodiment of the
10 invention. All amounts in ppm are on a weight/volume (g/ml) basis.
Moreover, the
application rates in brackets above are derived for a treatment utilizing a
standard
volume of 100 gallons of the specified solutions dispersed over 1 acre.
In yet other embodiments, the plant, in addition to being treated with
GABA, may also be treated with a composition that includes GABA and a GABA
15 agonist. For example, plants may be treated with baclofen as well as other
GABA
agonists known to the art, including, for example, cis-4-aminopent-2-enoic
acid
(CACA), imidazole-4-acetic acid (IAA) and 4,5,6,7-tetrahydroisoxazolo[5,4-
c]pyridin-3-of (THIP). Plants may also be treated with only a GABA antagonist,
such as picrotoxin or bicuculline, or only a GABA agonist to regulate plant
20 metabolism as desired. The plants may also be treated only with an agonist
or
antagonist of a benzodiazepine receptor, such as an animal peripheral
benzodiazepine receptor. Such compounds include quinine and spermine, and
other benzodiazepine receptor antagonists and agonists described herein.
GABA, the GABA agonists or antagonists and other agonists and
antagonists described herein are typically applied to the foliage of the plant
but
may also be administered as a soil drench. Furthermore, when plants are grown
hydroponically, the compounds and compositions may be applied to the aqueous
solution in which the plants are grown. The compositions are further
preferably
applied by spraying. Moreover, the compounds and compositions may also be
applied as a seed treatment.
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GABA, the GABA agonists or GABA antagonists, and other agonists and
antagonists described herein are preferably combined with a carrier medium as
known in the art. The compounds and compositions may, for example, be
combined with water, such as tap water or with distilled water to which has
been
added selected minerals. Alternatively, the compositions of the present
invention
may be applied as a solid. In such a form, the solid is preferably applied to
the
soil. The compositions may further include agricultural additives or
formulation
aids known to those skilled in the art. Such additives or aids may be used to
ensure
that the compositions disperse well in a spray tank, stick to or penetrate
plant
surfaces (particularly leaf or other foliage surfaces) as well as provide
other
benefits to the plant. For example, surfactants, dispersants, humectants, and
binders
may be used to disperse the compounds or compositions described herein in a
spray tank as well as to allow the compound or compositions to adhere to
and/or
penetrate the plant surfaces.
Methods of regulating plant metabolism are also provided by the present
invention. Regulation of plant metabolism may include positively or negatively
affecting nutrient utilization, such as nitrogen-assimilation, plant growth,
plant
productivity and the plant's resistance to the effects of plant stress. For
example,
in one form, an inventive method that may negatively affect plant productivity
includes introducing into a plant cell an antisense nucleotide sequence having
a
sequence complementary to a coding nucleotide sequence provided herein.
Accordingly, this invention also provides strategies for manipulating a gene
involved in plant receptor protein production and thus is an invaluable tool
for
further research of cellular stress and/or developmental processes. For
example,
manipulation of a plant receptor protein gene can provide quantitative
information
on the role of GABA-related processes on metabolic fluxes, .nutrient
utilization and
storage, cellular differentiation, growth, senescence, and signaling. Such
manipulation also provides a method for increasing crop productivity through
enhancing crop resistance to biotic and abiotic stresses. Crop quality and
yield is
improved by increasing tolerance to a variety of environmental stresses,
including
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disease, which cause a decrease in photosynthetic and nitrogen efficiency of
crop
plants resulting in decreased yields.
In one embodiment, the invention provides an antisense nucleotide
sequence that is complementary to a nucleotide sequence having at least about
50% identity to a length of nucleotides within the nucleotide sequence set
forth in
SEQ ID NO: 1. In another embodiment, the invention provides an antisense
nucleotide sequence that is complementary to a nucleotide sequence having at
least
about 60% identity to a length of nucleotides within the nucleotide sequence
set
forth in SEQ m NO: 1. In another embodiment, the invention provides an
to antisense nucleotide sequence that is complementary to a nucleotide
sequence
having at least about 70% identity to a length of nucleotides within the
nucleotide
sequence set forth in SEQ m NO: 1. In another embodiment, the invention
provides an antisense nucleotide sequence that is complementary to a
nucleotide
sequence having at least about SO% identity to a length of nucleotides within
the
15 nucleotide sequence set forth in SEQ )D NO: 1. In another embodiment, the
invention provides an antisense nucleotide sequence that is complementary to a
nucleotide sequence having at least about 90% identity to a length of
nucleotides
within the nucleotide sequence set forth in SEQ m NO: 1.
In one embodiment, the antisense nucleotide has a length of about 30 to
2o about 100 nucleotides. In another embodiment, the antisense nucleotide has
a
length of about 30 to about 200 nucleotides. In another embodiment, the
antisense
nucleotide has a length of about 30 to about 300 nucleotides. In another
embodiment, the antisense nucleotide has a length of and about 30 to about 400
nucleotides. In another embodiment, the antisense nucleotide sequence is as
long
25 as the entire length of the nucleotide sequence set forth in SEQ >D NO: 1.
The
antisense nucleotide sequence may hybridize to the template strand, which
serves
as the strand from which RNA is produced, so that transcription will be
reduced.
Alternatively, the antisense nucleotide sequence may be complementary to, and
therefore hybridize to, the RNA sequence, such as the mRNA sequence,
3o transcribed from the nucleotide sequences described herein, so that
translation of
the mRNA sequence to express the encoded protein will be reduced. The
antisense
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nucleotide sequence may be either DNA or RNA. Preferred antisense
oligonucleotides are complementary to the coding region of a particular
polynucleotide, although the sequences may in addition bind to selected
sequences
in a non-coding region. In further preferred forms of the invention, the
antisense
oligonucleotides will bind to nucleotides adjacent to the ATG initiation
codon.
In another form of the invention, a method is provided for regulating plant
metabolism by in vivo mutagenesis of the gene present in the plant genome
encoding the plant receptor protein described herein in order to alter its
activity to
provide the desired positive or negative results as described above. A plant
may be
l0 mutated by methods known to the skilled artisan, including chemical methods
and
DNA-insertion activation-tagged mutagenesis.
In.another aspect of the invention, methods of modifying receptor activity
in a plant are provided. In one form of the invention, a method includes
introducing into a plant cell a nucleic acid molecule having a nucleotide
sequence
encoding a plant protein as described herein.
In yet another aspect of the invention, methods of expressing plant proteins
expected to function as benzodiazepine receptors as described above are
provided.
In one embodiment, the method includes providing a nucleotide sequence
described above, or variants thereof, that encodes a protein described herein,
and
introducing the nucleotide sequence into a host cell, as described above. The
desired nucleotide sequence may be advantageously incorporated into a vector
to
form a recombinant vector. The recombinant vector may then be introduced into
a
host cell according to known procedures in the art. Such host cells are then
cultured under conditions, well known to the skilled artisan, effective to
achieve
expression of the plant protein. The protein may then be purified using
conventional techniques.
A wide variety of target plants are contemplated in accordance with the
invention. In one embodiment, the target plant is selected from the group
consisting of duckweed, rice, wheat, barley, rye, corn, Bermuda grass, Blue
grass,
3o fescue, rapeseed, potato, carrot, sweet potato, bean, pea, chicory,
lettuce, cabbage,
cauliflower, broccoli, turnip, radish, spinach, asparagus, onion, garlic,
eggplant,
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pepper, celery, squash, pumpkin, zucchini, cucumber, apple, pear, quince,
melon,
plum, cherry, peach, nectarine, apricot, strawberry, grape, raspberry,
blackberry,
pineapple, avocado, papaya, mango, banana, soybean, bush beans, tobacco,
tomato, green pepper, sorghum and sugarcane.
Any experiments, experimental examples, or experimental results provided
herein are intended to be illustrative of the present invention and should not
be
considered limiting or restrictive with regard to the invention scope.
Further, any
theory, mechanism of operation, or finding stated herein is meant to further
enhance
understanding of the present invention and is not intended to limit the
present
i0 invention in any way to such theory, mechanism or finding. All
publications,
patents, and patent applications cited in this specification are herein
incorporated by
reference as if each individual publication, patent, or patent application
were
specifically and individually indicated to be incorporated by reference and
set forth
in its entirety herein. While the invention has been illustrated and described
in
detail in the drawings and foregoing description, the same is to be considered
as
illustrative and not restrictive in character, it being understood that only
selected
embodiments have been shown and described and that all changes, equivalents,
and
modifications that come within the spirit of the invention described herein or
defined by the following claims are desired to be protected.
2o Reference will now be made to specific examples illustrating the invention
described above. It is to be understood that the examples are provided to
illustrate
preferred embodiments and that no limitation to the scope of the invention is
intended thereby.
EXAMPLE 1
Effect of Agonists and Antagonists of Animal Mitochondria) Benzodiazepine
Receptor on GABA-Mediated Growth Promotion in Duckweed
Benzodiazepine receptors are sensitive to the agonist diazepam and the
3o antagonists PK11195 (isoquinoline carboxamide), spermine, quinine and
cyclosporin A.
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Duckweed (LeYnha Minor L) was grown following the general procedure
described by Kinnersley (U.S. Patent No. 4,813,997) except that the culture
media
was Solu-Spray 20-20-20 fertilizer dissolved in tap water at 1 gll and the pH
was
adjusted to 5.5 as discussed in U.S. Patent No. 5,439,873 to Kinnersley.
5 Duckweed was treated with, independently, the indicated concentrations of
GABA
and either cyclosporin A, spermine, quinine, diazepam or PKl 1195.
As seen in FIGS. 2, 3, and 4, respectively, when duckweed was treated
independently with cyclosporin A, spermine, or quinine, in the presence of
GABA
in the medium, an inhibitory effect on growth was seen. Cyclosporin A is an
to immunosuppressant and has been shown to be the most potent pharmacological
inhibitor of the PTP in animal mitochondria. The inhibitory activity of
cyclosporin
has been attributed to binding to mitochondrial cyclophilin in the
mitochondrial
inner membrane. In duckweed experiments, 3 pM cyclosporin A significantly
inhibited plant growth in cultures containing 10 mM GABA. Relative to the
15 respective controls, inhibition of GABA-mediated growth by cyclosporin A
(FIG.
2), spermine (FIG. 3) and quinine (FIG. 4) was paradoxically greatest at
highest
levels of GABA. This is seen most clearly in FIG. 3, where dry weights in
cultures
with 10 mM GABA and 150 p,M spermine was significantly less than cultures
containing 150 ~uM spermine without any GABA. Addition of the benzodiazepine
2o diazepam at 3 p,M to cultures increased GABA-mediated growth (FIG. 5). The
increase in growth was significant at P < 0.05. The effect of diazepam on GABA
activity in plants is further evidence of structural similarity of GABA
receptors in
animals and plants.
Additionally, when duckweed was treated with PKl l 195, in the absence of
25 GABA, an inhibitory effect on growth was seen as shown in Table 1 below.
PK11195 is a diagnostic ligand of the peripheral benzodiazepine receptor,
which is
associated with the PTP in animal mitochondria. PKl 1195 blocked GABA-
mediated growth response at 50 p,M (FIG. 5).
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Table 1. Effect of PK11195 on dry weight of duckweed
Treatment Avg. Dry Weight of Duckweed
SD*
Control 33.2 2.9
PK11195 (50p,M) 34.4 5.6
PK11195 (100 p,M) 25.7 3.1
*Standard Deviation
The data in Table 1 suggests that when low levels of GABA are present in a
plant,
such as endogenous GABA levels, a higher concentration of PK1195 is needed to
see an effect.
As seen in FIG. 5, when duckweed, grown as above, was treated
independently with diazepam, an excitatory effect on growth was seen in the
presence
of GABA in the medium. When duckweed was treated independently with PKl 1195,
to an inhibitory effect on growth was seen in the presence of GABA in the
medium.
An earlier study reported the effects of antagonists of animal GABA
receptors on duckweed growth; however, the inhibitors of GABA bioactivity
reported in Table 2 below were up to 1000-fold more active than the GABA
receptor antagonists used in the earlier study. This suggests that they are
acting on
a different class of GABA receptors and that plants, like animals, likely have
a
multiplicity of GABA receptors.
Table 2. Effect of pharmacological agents on activity of the mitochondria)
permeability transition pore (PTP) and peripheral benzodiazepine receptor
(PBR)
in animals and on GABA-mediated growth activity in Lemna.
Action PTP/PBR (~.M) Lernna (wM)
Cyclosporin Inhibit 0.1 to 10.0 3 to 30
Spermine Inhibit 20 to 100 100 to 200
Quinine Inhibit 1.4 mM 100 to 400
Diazepam* Activate 10 to 100 3
PK11195* Inhibit 50 to 100 50
*Activity on animal mitochondria) benzodiazepine receptors
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The above results, taken together, provide evidence of benzodiazepine, or
benzodiazepine-like, receptors in plants, as experiments with chemicals that
promote or inhibit the activity of benzodiazepine receptors in animals have a
similar response in plants.
EXAMPLE 2
Isolation of a Full-length cDNA and Genomic DNA
Protocol
Arabidopsis thalia~ca (L.) Heynh. Ecotype Columbia (Col-0) seeds can be
to obtained from the Arabidopsis Biological Resource Center (Ohio State
University,
Columbus, OH). Arabidopsis seedlings are grown under aseptic conditions in
flasks
containing MS media [Murashige and Skook, Physiol. Plant 15:485 ( 1962)] on a
rotary
shaker (150 rpm). Two-day-old seedlings are collected for total RNA isolation.
Total
RNA are isolated as described in Turano, F.J. et a1.(1992) Plant Physiol.
100:374.
15 Primers, 5'EcoPBR(5'-GCCCGAATTCATGGCCGAGACAGAGAGGAAAAGC-3')
and 3'EcoPBR (5'-GCCCGAATTCTCACGCGACTGCAAGCTTTACATT -3') (SEQ
ID NOS: 3 and 4, respectively) (corresponding to GenBank, unknown protein,
gene #
At2g47770, protein id=AAC63632.1, db xref--GI: 3738290) are commercially
synthesized (Biosynthesis, Inc., Lewisville, TX) and used for RT-PCR
reactions. For
20 the RT-PCR, a 5' RACE system (Life Technologies, Rockville, MD) is used to
identify
a full-length cDNA clone. Primer 3'EcoPBR is used to synthesize a first strand
cDNA
from 1 ~.g of poly (A+)RNA isolated from two-day-old plants following the
manufacturers instructions. One-fifth of the first strand cDNA synthesis is
used as a
template in a gene amplification reaction with both primers, 5'EcoPBR and
3'EcoPBR.
25 Prior to the amplification, the components are incubated at 95°C for
4 minutes. The
gene amplification reaction is conducted at 94°C for 1 minute,
68°C for 1 minute and
72°C for 2 minutes, for 30 cycles followed by a 5 minute, 72°C
extension.
Genomic DNA is isolated from leaves of 24 day old Arabidopsis as
described in Turano, F.J. et al. ( 1992) Plant Physiol 100:374. For the PCR
3o reaction, 250 ng of each primer (5'EcoPBR and 3'EcoPBR) is used with
approximately 500 ng of genomic DNA. Prior to the amplification reaction, the
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components are incubated at 95°C for 10 minutes. The gene amplification
reaction
is conducted at 94°C for 1 minutes, 70°C for 1 minute and
72°C for 3 minutes, for
30 cycles followed by a 5 minute, 72°C extension.
Both the genomic DNA and cDNA fragments are cloned separately into
PCR2.1 (Invitrogen Corp. Carlsbad, CA, USA) and sequenced using the Taq
Dideoxy terminator cycle sequence (Applied Biosystems) method. The data is
analyzed with MacVector software on a Power Macintosh 6500/250.
EXAMPLE 3
Construction of a Transgenic Plant
A transgenic plant that overexpresses a plant receptor protein, or one that
overexpresses an antisense receptor protein is made as follows. The entire
(591
base pairs) open reading frame for the sense (over-expression) or antisense
(under-
expression) of the receptor protein, or the portions thereof as small as about
25
base pairs (for antisense or RNAi only), is cloned into a plant transformation
vector, such as pBIl21(Clonetech, Palo Alto, CA) using PCR, RT-PCR or
conventional cloning methods to make antisense constructs. Gene specific
primers,
5'EcoPBR(5'-GCCCGAATTCATGGCCGAGACAGAGAGGAAAAGC-3') and
3'EcoPBR (5'-GCCCGAATTCTCACGCGACTGCAAGCTTTACATT -3')
(corresponding to GenBank, unknown protein, gene # At2g47770,
protein id=AAC63632.1, db_xref--GI: 373290) are commercially synthesized
(Biosynthesis Inc., Lewisville, TX, USA) and used for PCR or RT-PCR reactions.
For example, the PCR reactions use 250 ng of each primer with approximately
500
ng of genomic DNA. Prior to the amplification reaction, the components are
incubated at 95°C for 2 min. The gene amplification reaction is
conducted at 94°C
for 1 min, 65°C for 1 min and 72°C for 2 min, for 30 cycles
followed by a 4 min
72°C extension.
For the RT-PCR, a 5' RACE system (Life Technologies, Rockville, MD,
USA) or a simpler reverse transcriptase (RT) based system, is used to identify
a
3o full-length cDNA clone. Primer 3'EcoPBR is used to synthesize first strand
cDNA
from 1 ~,g from poly (A+) RNA isolated from 2 day old plants following the
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manufacturer's instructions. One fifth of the first strand cDNA synthesis is
used as
a template in a gene amplification reaction with both primers, 5'EcoPBR and
3'EcoPBR. Prior to the amplification, the components are incubated at
95°C for 2
min. The gene amplification reaction is conducted at 94°C for 1 min,
58°C for 1
min and 72°C for 2 min, for 30 cycles followed by a 5 min 72°C
extension.
The genomic DNA or cDNA fragments are cloned into plant transformation
vectors in a sense (forward) or anti sense (backwards) direction, depending on
the
desired result. The vectors may contain constitutive promoters such as CaMV
35S
promoter and the nopaline synthase terminator, or other promoters described
herein
and known to the art. The vectors may be modified to include promoters that
can be
induced by biotic [Sohal et a1.,(1999) Plant Mol. Biol. 41:75-87] or abiotic
stresses
[Ngai et al., (1997) Plant J. 12:1021-1034; van Der Krol et al., (1999) Plant
Physiol. 121:1153-1162; Kucho et al., (1999) PlantPhysiol 121:1329-1338]
and/or
hormones and other signaling molecules [Chen and Singh, (1999) Plant J. 19:667-
677; Lu et al., (1998) J. Biol. Chem. 273:10120-10131; Leubner-Metzger et al.,
(1998) PlantMol. Biol. 38:785-795]. The orientation of the cloned constructs
is
confirmed by restriction endonuclease and PCR analyses.
Upon completion of cloning, the binary vector construct is transferred into
a disarmed strain of Agrobacterium to»zefaciens, such as EHA105, and
subsequently into Arabidopsis (Ws ecotype) using the vacuum infiltration
method
[Bechtold, N. and Bouchez, D. (1995) Ire planta Agrobacteriurn-mediated
transformation of adult Arabidopsis thaliana plants by vacuum infiltration. In
Gene
Transfer to Plants. I. Potrykus and G. Spangenberg Eds. Springer-Verlag,
Heidelberg, pp. 19-23] with one modification (i.e., the addition of 0.02%
(v/v)
Silwet to the infiltration media). Seeds collected from the transformed plants
are
germinated and selected for kanamycin resistance.
While the invention has been illustrated and described in detail in the
figures and foregoing description, the same is to be considered as
illustrative and
not restrictive in character, it being understood that only the preferred
embodiment
has been shown and described and that all changes and modifications that come
within the spirit of the invention are desired to be protected. In addition,
all
CA 02453428 2004-O1-19
WO 03/007886 PCT/US02/23180
references cited herein are indicative of the level of skill in the art and
are hereby
incorporated by reference in their entirety.
CA 02453428 2004-O1-19
WO 03/007886 PCT/US02/23180
SEQUENCE LISTING
<110> Kinnersley, Alan M.
Turano, Frank ,7.
<120> Plant Ion Channels and Methods
<130> 7224-57
<140> N/A
<141> 2001-07-20
<160> 4
<170> PatentIn version 3.1
<210> 1
<211> 591
<212> DNA
<213> Arabidopsis thaliana
<220>
<221> CDS
<222> (1)..(591)
<223>
<400> 1
atg gat tct cag gac atc aga tac cgc ggc gga gac gac aga gac get 48
Met Asp Ser Gln Asp Ile Arg Tyr Arg Gly Gly Asp Asp Arg Asp Ala
1 5 10 15
gca acg acg get atg gcc gag aca gag agg aaa agc get gac gac aac 96
Ala Thr Thr Ala Met Ala Glu Thr Glu Arg Lys Ser Ala Asp Asp Asn
20 25 30
aaa gga aaa cgc gat caa aag agg gcg atg gcg aaa cgt ggt ctc aag 144
Lys Gly Lys Arg Asp Gln Lys Arg Ala Met Ala Lys Arg Gly Leu Lys
35 40 ~ 45
tct ctg acg gta gcg gtt gcg get cct gtg ctc gtg acg ctc ttc get 192
Ser Leu Thr Val Ala Val Ala Ala Pro Val Leu Val Thr Leu Phe Ala
50 55 60
acg tat ttc ctc ggc aca agc gac gga tac ggg aat cga get aag tcc 240
Thr Tyr Phe Leu Gly Thr Ser Asp Gly Tyr Gly Asn Arg Ala Lys Ser
65 70 75 80
tcg tcg tgg atc cca cct ctg tgg ctc cta cac aca acg tgt ctc get 288
Ser Ser Trp Ile Pro Pro Leu Trp Leu Leu His Thr Thr Cys Leu Ala
85 90 95
tct agt ggt ctg atg ggt ttg get gcg tgg ctt gta tgg gtt gac ggt 336
Ser Ser Gly Leu Met Gly Leu Ala Ala Trp Leu Val Trp Val Asp Gly
100 105 110
ggc ttc cac aag aag ccc aat get ctg tat ctt tac tta get cag ttt 384
1/3
CA 02453428 2004-O1-19
WO 03/007886 PCT/US02/23180
Gly Phe His Lys Lys Pro Asn Ala Leu Tyr Leu Tyr Leu Ala Gln Phe
115 120 125
ttg~ctctgtttg gtttgg gatccggtt acgttccgc gtcgggtcg gga 432
LeuLeuV Leu al Trp AspProVal ThrPheArg ValGlySer Gly
Cys
13 13 140
0 5
gtagcggggctt gcggtg tggttgggt caatcgget gcgttattc gga 480
ValAlaGlyLeu AlaVal TrpLeuG1y GlnSerAla AlaLeuPhe Gly
145 150 155 160
tgctacaaggcc tttaat gagataagt ccggtcget ggtaatctg gta 528
CysTyrLysAla PheAsn GluIleSer ProValAla GlyAsnLeu Va1
165 l70 175
aagccgtgtttg gettgg getgccttt gtagccget gttaatgta aag 576
LysProCysLeu AlaTrp AlaAlaPhe ValA1aAla ValAsnVal Lys
180 185 190
cttgcagtcgcg tga 591
LeuAlaValAla
195
<210> 2
<211> 196
<212> PRT
<213> Arabidopsis thaliana
<400> 2
Met Asp Ser Gln Asp Ile Arg Tyr Arg Gly Gly Asp Asp Arg Asp Ala
1 5 10 15
Ala Thr Thr Ala Met Ala Glu Thr Glu Arg Lys Ser Ala Asp Asp Asn
20 25 30
Lys Gly Lys Arg Asp Gln Lys Arg Ala Met Ala Lys Arg Gly Leu Lys
35 40 45
Ser Leu Thr Val Ala Val Ala A1a Pro Val Leu Val Thr Leu Phe Ala
50 55 60
Thr Tyr Phe Leu Gly Thr Ser Asp Gly Tyr Gly Asn Arg Ala Lys Ser
65 70 75 80
Ser Ser Trp Ile Pro Pro Leu Trp Leu Leu His Thr Thr Cys Leu Ala
85 90 95
Ser Ser Gly Leu Met Gly Leu A1a Ala Trp Leu Val Trp Val Asp Gly
100 105 110
2/3
CA 02453428 2004-O1-19
WO 03/007886 PCT/US02/23180
Gly Phe His Lys Lys Pro Asn Ala Leu Tyr Leu Tyr Leu Ala Gln Phe
115 120 125
Leu Leu Cys Leu Val Trp Asp Pro Val Thr Phe Arg Val Gly Ser Gly
130 135 140
Val Ala Gly Leu Ala Val Trp Leu Gly Gln Ser Ala Ala Leu Phe Gly
145 150 155 160
Cys Tyr Lys Ala Phe Asn G1u 21e Ser Pro Val Ala Gly Asn Leu Val
165 170 175
Lys Pro Cys Leu Ala Trp Ala Ala Phe Val Ala Ala Val Asn Val Lys
180 185 190
Leu Ala Val Ala
195
<210> 3
<211> 34
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for PCR Reaction described in Example 2
<400> 3
gcccgaattc atggccgaga. cagagaggaa aagc 34
<210> 4
<211> 34
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for PCR reaction described in Example 2
<400> 4
gcccgaattc tcacgcgact gcaagcttta catt 34
3/3
